Particularly in live cell microscopy, the exact refractive index of the specimen is usually unknown. This relates not (only) to the embedding medium and/or an employed buffer but, in particular, to the interior of the specimen, which may be provided, for example, by cells, (tumour) spheroids, tissue slices or entire organisms.
Since these specimens are usually stored and examined in aqueous solutions (buffer solutions), and for historical reasons, water immersion objectives are usually used. These are optimized for a refractive index of n=1.33 (refractive index of water). However, the refractive index of living cell specimens deviates from n=1.33 (see, e.g., Liu, P. Y., et al. “Cell refractive index for cell biology and disease diagnosis: past, present and future.” Lab on a Chip 16.4 (2016): 634-644).
Thus, for example, the refractive index of the cytosol of typical cells lies in the range of 1.36-1.39 and therefore deviates significantly from the refractive index of water. A maladjustment of optical arrangements resulting therefrom leads to aberrations which restrict the optical power of the microscope. A compensation of these aberrations, if at all possible, is complicated and expensive and can be brought about, for example, by means of adaptive optical units, e.g., deformable mirrors.
This applies, in particular, to light sheet systems, which have at least one optical axis that is not perpendicular to a coverslip. The coverslip, which may also be formed by the base of a specimen holder, for example a Petri dish, a (micro-)titration plate or a transparent plate in the case of inverted microscopy, is passed through at an angle not equal to 90°, i.e., passed through obliquely, by detection radiation to be captured.
When errors occur in the adaptation of the refractive index, non-symmetrical aberrations such as astigmatism or coma often arise. However, these are even less tolerable than symmetrical aberrations which may occur in the case of conventional arrangements with reflected light and which appear, in particular, as spherical aberrations. Although such symmetric errors are initially more easily tolerable by the user, they ultimately nevertheless lead to significant restriction, for example in the optical penetration depth of the microscope.
Suitable immersion media can be used for reducing aberrations that are caused during a passage of an illumination radiation and/or detection radiation through a specimen.
Conventional immersion media include, for example, water, silicone oil and immersion oils that are used together with a correspondingly embodied immersion objective. In microscopy, the use of immersion objectives offers many advantages, which all ultimately arise from the higher obtainable numerical apertures of the objectives. These advantages include, for example, a higher spatial resolution, a higher light collection efficiency, an improved signal-to-noise or signal-to-background ratio, shorter exposure times and a better temporal resolution and reduced phototoxicity.
Since the specimens can be embedded and affixed differently, there are different classes of immersion media for working as closely as possible to the refractive index of the specimen. Typical immersion media include water, organic substitute media for water (e.g., ZEISS Immersol W), glycerol (e.g., ZEISS Immersol G) and special immersion oils (e.g., ZEISS Immersol F, Immersol HI). All these immersion media are liquid at room temperatures. Ideal image quality, maximum signal intensity and maximum penetration depth are only obtained if the immersion medium has exactly the same optical parameters as the specimen (cf., Hell, S., et al. (1993): Aberrations in confocal fluorescence microscopy induced by mismatches in refractive index. Journal of microscopy, 169: 391-405).
In principle, the numerical aperture can be maximized by using immersion media with a refractive index that is as high as possible, with an expedient upper limit being given by the refractive index of the coverslip. The coverslip is no longer optically effective in the case of a perfectly adapted refractive index (nimmersion=ncoverslip). Such a situation, which is typically obtained in the case of oil immersion objectives, is illustrated schematically in FIG. 1a. 
A specimen 14, a specimen holder 11 in the form of a coverslip and an objective 10 and also an immersion medium 6 between specimen holder 11 and objective 10 are illustrated. The profile of focussed illumination radiation is illustrated in a simplified manner as an interrupted solid line.
A case often occurring in the case of biological specimens is that the refractive index of the specimen medium deviates from that of the coverslip 11 and the immersion. In this situation, ideal work can only be carried out relatively close to the coverslip surface when using oil immersion objectives. The reason for this lies in the fact that aberrations, in particular spherical aberrations, occur as a result of the refractive index jump, said aberrations becoming ever stronger with increasing penetration into the specimen 14. Therefore, water immersion objectives (FIGS. 1b and 1c) are better suited to microscopy of living cells, in particular. Since the cells are in an aqueous solution, the refractive indices of immersion liquid and specimen medium are very similar; there is only a deviation of the coverslip 11 itself. If the objective has been optically corrected, no spherical aberrations occur in the case of relatively deep penetration into the specimen 14 (FIG. 1c). However, this correction only works for a known and specified coverslip thickness and glass type. Therefore, water immersion objectives usually have a correction function, by means of which unavoidable coverslip thickness variations, as indicated by the double-headed arrow (FIGS. 1b and 1c), can be corrected by displacing a lens or lens group in the objective 10 (correction ring, “Corr ring”).